Proofreading (biology)

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The term proofreading is used in genetics to refer to the error-correcting processes, first proposed by John Hopfield and Jacques Ninio, involved in DNA replication, immune system specificity, and enzyme-substrate recognition among many other processes that require enhanced specificity. The proofreading mechanisms of Hopfield and Ninio are non-equilibrium active processes that consume ATP to enhance specificity of various biochemical reactions.

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In bacteria, all three DNA polymerases (I, II and III) have the ability to proofread, using 3’ → 5’ exonuclease activity. When an incorrect base pair is recognized, DNA polymerase reverses its direction by one base pair of DNA and excises the mismatched base. Following base excision, the polymerase can re-insert the correct base and replication can continue.

In eukaryotes, only the polymerases that deal with the elongation (delta and epsilon) have proofreading ability (3’ → 5’ exonuclease activity). [1]

Proofreading also occurs in mRNA translation for protein synthesis. [2] In this case, one mechanism is the release of any incorrect aminoacyl-tRNA before peptide bond formation. [3]

The extent of proofreading in DNA replication determines the mutation rate, and is different in different species. [4] For example, loss of proofreading due to mutations in the DNA polymerase epsilon gene results in a hyper-mutated genotype with >100 mutations per Mbase of DNA in human colorectal cancers. [5]

The extent of proofreading in other molecular processes can depend on the effective population size of the species and the number of genes affected by the same proofreading mechanism. [6]

Bacteriophage T4 DNA polymerase

Bacteriophage (phage) T4 gene 43 encodes the phage’s DNA polymerase replicative enzyme. Temperature-sensitive (ts) gene 43 mutants have been identified that have an antimutator phenotype, that is a lower rate of spontaneous mutation than wild type. [7] Studies of one of these mutants, tsB120, showed that the DNA polymerase specified by this mutant copies DNA templates at a slower rate than the wild-type polymerase. [8] However, the 3’ to 5’ exonuclease activity was no higher than wild-type. During DNA replication the ratio of nucleotides turned over to those stably incorporated into newly formed DNA is 10 to 100 times higher in the case of the tsB120 mutant than in wild-type. [8] It was proposed that the antimutator effect may be explained by both greater accuracy in nucleotide selection and an increased efficiency of removal of noncomplementary nucleotides (proofreading) by the tsB120 polymerase.

When phage T4 virions with a wild-type gene 43 DNA polymerase are exposed to either ultraviolet light, which introduces cyclobutane pyrimidine dimer damages in DNA, or psoralen-plus-light, which introduces pyrimidine adducts, the rate of mutation increases. However, these mutagenic effects are inhibited when the phage's DNA synthesis is catalyzed by the tsCB120 antimutator polymerase, or another antimutator polymerase, tsCB87. [9] These findings indicate that the level of induction of mutations by DNA damage can be strongly influenced by the gene 43 DNA polymerase proofreading function.

SARS-CoV-2 proofreading enzyme

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 pandemic. The SARS-CoV-2 RNA virus genome encodes a replication-and transcription complex, a multisubunit protein machine that carries out viral genome replication and transcription, processes essential to the virus life cycle. One of the proteins specified by the coronavirus genome is a non-structural protein, nsp14, that is a 3’-to-5’ exoribonuclease (ExoN). This protein resides in the protein complex nsp10-nsp14 that enhances replication fidelity by proofreading RNA synthesis, an activity critical for the virus life cycle. [10] Furthermore the coronavirus proofreading exoribonuclease nsp14-ExoN is required for maintaining genetic recombination generated during infection. [11]

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<span class="mw-page-title-main">Lambda phage</span> Bacteriophage that infects Escherichia coli

Enterobacteria phage λ is a bacterial virus, or bacteriophage, that infects the bacterial species Escherichia coli. It was discovered by Esther Lederberg in 1950. The wild type of this virus has a temperate life cycle that allows it to either reside within the genome of its host through lysogeny or enter into a lytic phase, during which it kills and lyses the cell to produce offspring. Lambda strains, mutated at specific sites, are unable to lysogenize cells; instead, they grow and enter the lytic cycle after superinfecting an already lysogenized cell.

<span class="mw-page-title-main">DNA polymerase</span> Form of DNA replication

A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create two identical DNA duplexes from a single original DNA duplex. During this process, DNA polymerase "reads" the existing DNA strands to create two new strands that match the existing ones. These enzymes catalyze the chemical reaction

<span class="mw-page-title-main">DNA polymerase I</span> Family of enzymes

DNA polymerase I is an enzyme that participates in the process of prokaryotic DNA replication. Discovered by Arthur Kornberg in 1956, it was the first known DNA polymerase. It was initially characterized in E. coli and is ubiquitous in prokaryotes. In E. coli and many other bacteria, the gene that encodes Pol I is known as polA. The E. coli Pol I enzyme is composed of 928 amino acids, and is an example of a processive enzyme — it can sequentially catalyze multiple polymerisation steps without releasing the single-stranded template. The physiological function of Pol I is mainly to support repair of damaged DNA, but it also contributes to connecting Okazaki fragments by deleting RNA primers and replacing the ribonucleotides with DNA.

dnaQ is the gene encoding the ε subunit of DNA polymerase III in Escherichia coli. The ε subunit is one of three core proteins in the DNA polymerase complex. It functions as a 3’→5’ DNA directed proofreading exonuclease that removes incorrectly incorporated bases during replication. dnaQ may also be referred to as mutD.

<i>Escherichia virus T4</i> Species of bacteriophage

Escherichia virus T4 is a species of bacteriophages that infect Escherichia coli bacteria. It is a double-stranded DNA virus in the subfamily Tevenvirinae of the family Straboviridae. T4 is capable of undergoing only a lytic life cycle and not the lysogenic life cycle. The species was formerly named T-even bacteriophage, a name which also encompasses, among other strains, Enterobacteria phage T2, Enterobacteria phage T4 and Enterobacteria phage T6.

DNA gyrase, or simply gyrase, is an enzyme within the class of topoisomerase and is a subclass of Type II topoisomerases that reduces topological strain in an ATP dependent manner while double-stranded DNA is being unwound by elongating RNA-polymerase or by helicase in front of the progressing replication fork. It is the only known enzyme to actively contribute negative supercoiling to DNA, while it also is capable of relaxing positive supercoils. It does so by looping the template to form a crossing, then cutting one of the double helices and passing the other through it before releasing the break, changing the linking number by two in each enzymatic step. This process occurs in bacteria, whose single circular DNA is cut by DNA gyrase and the two ends are then twisted around each other to form supercoils. Gyrase is also found in eukaryotic plastids: it has been found in the apicoplast of the malarial parasite Plasmodium falciparum and in chloroplasts of several plants. Bacterial DNA gyrase is the target of many antibiotics, including nalidixic acid, novobiocin, albicidin, and ciprofloxacin.

<span class="mw-page-title-main">Exonuclease</span> Class of enzymes; type of nuclease

Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3′ or the 5′ end occurs. Its close relative is the endonuclease, which cleaves phosphodiester bonds in the middle (endo) of a polynucleotide chain. Eukaryotes and prokaryotes have three types of exonucleases involved in the normal turnover of mRNA: 5′ to 3′ exonuclease (Xrn1), which is a dependent decapping protein; 3′ to 5′ exonuclease, an independent protein; and poly(A)-specific 3′ to 5′ exonuclease.

<span class="mw-page-title-main">Ethyl methanesulfonate</span> Chemical compound

Ethyl methanesulfonate (EMS) is an organosulfur compound with the formula CH3SO3C2H5. It is the ethyl ester of methanesulfonic acid. A colorless liquid, it is classified as an alkylating agent. EMS is the most commonly used chemical mutagen in experimental genetics. Mutations induced by EMS exposure can then be studied in genetic screens or other assays.

<span class="mw-page-title-main">T7 phage</span> Species of virus

Bacteriophage T7 is a bacteriophage, a virus that infects bacteria. It infects most strains of Escherichia coli and relies on these hosts to propagate. Bacteriophage T7 has a lytic life cycle, meaning that it destroys the cell it infects. It also possesses several properties that make it an ideal phage for experimentation: its purification and concentration have produced consistent values in chemical analyses; it can be rendered noninfectious by exposure to UV light; and it can be used in phage display to clone RNA binding proteins.

<span class="mw-page-title-main">DNA adenine methylase</span> Prokaryotic enzyme

DNA adenine methylase, (Dam) (also site-specific DNA-methyltransferase (adenine-specific), EC 2.1.1.72, modification methylase, restriction-modification system) is an enzyme that adds a methyl group to the adenine of the sequence 5'-GATC-3' in newly synthesized DNA. Immediately after DNA synthesis, the daughter strand remains unmethylated for a short time. It is an orphan methyltransferase that is not part of a restriction-modification system and regulates gene expression. This enzyme catalyses the following chemical reaction

<span class="mw-page-title-main">RNA-dependent RNA polymerase</span> Enzyme that synthesizes RNA from an RNA template

RNA-dependent RNA polymerase (RdRp) or RNA replicase is an enzyme that catalyzes the replication of RNA from an RNA template. Specifically, it catalyzes synthesis of the RNA strand complementary to a given RNA template. This is in contrast to typical DNA-dependent RNA polymerases, which all organisms use to catalyze the transcription of RNA from a DNA template.

A nonsense suppressor is a factor which can inhibit the effect of the nonsense mutation. Nonsense suppressors can be generally divided into two classes: a) a mutated tRNA which can bind with a termination codon on mRNA; b) a mutation on ribosomes decreasing the effect of a termination codon. It is believed that nonsense suppressors keep a low concentration in the cell and do not disrupt normal translation most of the time. In addition, many genes do not have only one termination codon, and cells commonly use ochre codons as the termination signal, whose nonsense suppressors are usually inefficient.

Temperature-sensitive mutants are variants of genes that allow normal function of the organism at low temperatures, but altered function at higher temperatures. Cold sensitive mutants are variants of genes that allow normal function of the organism at higher temperatures, but altered function at low temperatures.

Lethal alleles are alleles that cause the death of the organism that carries them. They are usually a result of mutations in genes that are essential for growth or development. Lethal alleles may be recessive, dominant, or conditional depending on the gene or genes involved.

<span class="mw-page-title-main">POLD1</span> Protein-coding gene in the species Homo sapiens

DNA polymerase delta catalytic subunit(DPOD1) is an enzyme that is encoded in the human by the POLD1 gene, in the DNA polymerase delta complex. DPOD1 is responsible for synthesizing the lagging strand of DNA, and has also been implicated in some activities at the leading strand. The DPOD1 subunit encodes both DNA polymerizing and exonuclease domains, which provide the protein an important second function in proofreading to ensure replication accuracy during DNA synthesis, and in a number of types of replication-linked DNA repair following DNA damage.

<span class="mw-page-title-main">T7 DNA polymerase</span> Enzyme

T7 DNA polymerase is an enzyme used during the DNA replication of the T7 bacteriophage. During this process, the DNA polymerase “reads” existing DNA strands and creates two new strands that match the existing ones. The T7 DNA polymerase requires a host factor, E. coli thioredoxin, in order to carry out its function. This helps stabilize the binding of the necessary protein to the primer-template to improve processivity by more than 100-fold, which is a feature unique to this enzyme. It is a member of the Family A DNA polymerases, which include E. coli DNA polymerase I and Taq DNA polymerase.

Φ29 DNA polymerase is an enzyme from the bacteriophage Φ29. It is being increasingly used in molecular biology for multiple displacement DNA amplification procedures, and has a number of features that make it particularly suitable for this application. It was discovered and characterized by Spanish scientists Luis Blanco and Margarita Salas.

John W. Drake was an American microbiologist and geneticist, working for over half a century in the field of mutagenesis and DNA repair.

Charles 'Charley' M. Steinberg was an immunobiologist and permanent member of the Basel Institute for Immunology. He was a former student of Max Delbrück. Notably he hosted Richard Feynman at Caltech when Feynman studied molecular biology, leading Feynman to remark that Charlie was “...the smartest guy I know”. He was instrumental in the discovery of V(D)J recombination, bacteriophage genetics as part of the phage group and co-discoverer of the amber-mutant of the T4 bacteriophage that led to the recognition of stop codons.

Charles Clifton Richardson is an American biochemist and professor at Harvard University. Richardson received his undergraduate education at Duke University, where he majored in medicine. He received his M.D. at Duke Medical School in 1960. Richardson works as a professor at Harvard Medical School, and he served as editor/associate editor of the Annual Review of Biochemistry from 1972 to 2003. Richardson received the American Chemical Society Award in Biological Chemistry in 1968, as well as numerous other accolades.

References

  1. ↑ Moldovan, G. L.; Pfander, B.; Jentsch, S. (2007). "PCNA, the Maestro of the Replication Fork". Cell. 129 (4): 665–679. doi: 10.1016/j.cell.2007.05.003 . PMID   17512402. S2CID   3547069.
  2. ↑ Pharmamotion --> Protein synthesis inhibitors: aminoglycosides mechanism of action animation. Classification of agents Archived 2010-03-12 at the Wayback Machine Posted by Flavio Guzmán on 12/08/08
  3. ↑ Translation: Protein Synthesis by Joyce J. Diwan. Rensselaer Polytechnic Institute. Retrieved October 2011 Archived 2016-03-07 at the Wayback Machine
  4. ↑ Drake, J. W.; Charlesworth, B; Charlesworth, D; Crow, J. F. (1998). "Rates of spontaneous mutation". Genetics. 148 (4): 1667–86. doi:10.1093/genetics/148.4.1667. PMC   1460098 . PMID   9560386.
  5. ↑ The Cancer Genome Atlas Network; Bainbridge; Chang; Dinh; Drummond; Fowler; Kovar; Lewis; Morgan; Newsham; Reid; Santibanez; Shinbrot; Trevino; Wu; Wang; Gunaratne; Donehower; Creighton; Wheeler; Gibbs; Lawrence; Voet; Jing; Cibulskis; Sivachenko; Stojanov; McKenna; Lander; et al. (2012). "Comprehensive molecular characterization of human colon and rectal cancer". Nature. 487 (7407): 330–7. Bibcode:2012Natur.487..330T. doi:10.1038/nature11252. PMC   3401966 . PMID   22810696.
  6. ↑ Rajon E, Masel J (2011). "Evolution of molecular error rates and the consequences for evolvability". PNAS. 108 (3): 1082–7. Bibcode:2011PNAS..108.1082R. doi: 10.1073/pnas.1012918108 . PMC   3024668 . PMID   21199946.
  7. ↑ Drake JW, Allen EF (1968). "Antimutagenic DNA polymerases of bacteriophage T4". Cold Spring Harb Symp Quant Biol. 33: 339–44. doi:10.1101/sqb.1968.033.01.039. PMID   5254574.
  8. 1 2 Gillin FD, Nossal NG (September 1976). "Control of mutation frequency by bacteriophage T4 DNA polymerase. I. The CB120 antimutator DNA polymerase is defective in strand displacement". J Biol Chem. 251 (17): 5219–24. doi: 10.1016/S0021-9258(17)33149-6 . PMID   956182.
  9. ↑ Yarosh DB, Johns V, Mufti S, Bernstein C, Bernstein H (April 1980). "Inhibition of UV and psoralen-plus-light mutagenesis in phage T4 by gene 43 antimutator polymerase alleles". Photochem Photobiol. 31 (4): 341–350. doi:10.1111/j.1751-1097.1980.tb02551.x. PMID   7384228.
  10. ↑ Liu C, Shi W, Becker ST, Schatz DG, Liu B, Yang Y (September 2021). "Structural basis of mismatch recognition by a SARS-CoV-2 proofreading enzyme". Science. 373 (6559): 1142–6. Bibcode:2021Sci...373.1142L. doi:10.1126/science.abi9310. PMC   9836006 . PMID   34315827.
  11. ↑ Gribble J, Stevens LJ, Agostini ML, Anderson-Daniels J, Chappell JD, Lu X, Pruijssers AJ, Routh AL, Denison MR (January 2021). "The coronavirus proofreading exoribonuclease mediates extensive viral recombination". PLOS Pathog. 17 (1): e1009226. doi: 10.1371/journal.ppat.1009226 . PMC   7846108 . PMID   33465137.


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